1. Field of the Invention
The present invention relates generally to a method for the generation of a substantially homogenous population of undifferentiated cells. More particularly, the present invention relates to a method for isolating a substantially homogenous population of stem cells, and in particular, mammary stem cells (MaSCs). The MaSCs of the present invention are isolated on the basis of differential levels of proteins present on their cell surface. The MaSCs of the present invention are particularly useful as targets for identifying agents which modulate MaSC survival, self-renewal, proliferation and/or differentiation in both normal and diseased tissue such as, but not limited to, tumor tissue, and, also as source of tissue for the regeneration, replacement and/or augmentation of tissue damaged and/or lost after disease or injury.
2. Description of the Prior Art
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Bibliographic details of references provided in this document are listed at the end of the specification.
Breast cancer is the most common malignancy to affect women, accounting for approximately one quarter of all female cancers. Despite a significant improvement in the management of breast cancer over the last few years, about 25% of women diagnosed will die from the disease, revealing that those tumor cells have intrinsic properties that are refractory to current treatment strategies. The heterogeneous nature of breast cancer suggests the involvement of multiple genetic factors and cell types but these are poorly understood.
A prerequisite to understanding breast oncogenesis is the study of the regulation of normal breast epithelial development. The mammary gland is composed of a branching network of ducts and lobuloaveolar structures, the latter arising through pregnancy. There are two primary epithelial cell lineages, myoepithelial and luminal (comprising ductal and alveolar subtypes), which are presumed to arise from a common progenitor cell referred to herein as a mammary stem cell or MaSC (for review see Smalley and Ashworth, Nat Rev Cancer 3:832-844, 2003). The concept of an organ-specific stem cell is well established for haematopoiesis, as well as other organ systems (e.g. Rietze et al., Nature 214:736-739, 2001; Li et al., Nat Med 9:1293-1299, 2003; Morris et al., Nat Biotech 22:411-417, 2004; Tumbar et al., Science 303:359-363, 2004). It has been hypothesized that stem and progenitor cells (also known as transit amplifying cells) are critical cellular targets during tumorigenesis, and that deregulated expression of genes normally expressed in mammary stem and progenitor cells contribute to the pathogenesis of breast cancer (Reya et al., Nature 414:105-111, 2001). The existence of a breast cancer “stem cell” may, in fact, be one explanation for resistance to existing anti-cancer drugs and eventual emergence of disease that is refractory to therapy (Al-Hajj et al., PNAS 100:3983-3988, 2004).
The mammary gland normally develops postnatally (at puberty), through a process of ductal elongation and branching that extends from the nipple region to penetrate the stromal tissue of the mammary gland (“the mammary fat pad” or MFP). This process is primarily driven by oestrogen and progesterone, and also requires prolactin. In the adult gland, the mammary gland is therefore comprised of the stomal elements and branching ducts. The ducts are comprised of luminal epithelial cells and surrounding myoepithelial cells, which are believed to arise from a common precursor cell. These are surrounded by a basement membrane. During pregnancy further development and functional maturation of the mammary gland occurs through additional ductal outgrowth and branching and the outgrowth of lobuloalveolar structures, which are the milk-secreting units in the fully differentiated gland. Lobuloalveolar units are comprised of alveolar epithelial and myoepithelial cells, and are also surrounded by a basement membrane. Following the cessation of lactation, the mammary gland undergoes a process of coordinated involution, whereby the lobuloalveolar units and some ducts regress through a process of programmed cell death and remodelling. This entire process undergoes repeated cycles with each pregnancy. Stem cells and progenitor cells are necessary for adult mammary gland development and the sequential rounds of epithelial cell development with each pregnancy cycle. It has been proposed that a resting stem cell undergoes coordinated lineage specification and commitment to pre-luminal or pre-myoepithelial progenitor cells, which in turn differentiate into functional ductal and alveolar luminal cells and myoepithelial cells, respectively (FIG. 1).
The existence of MaSCs has been confirmed through serial transplantation studies using epithelial mammary explants in mice (Daniel et al., PNAS 61:53-60, 1968). This technique involves transfer of small donor mammary explants into the de-epithelialized MFPs of pre-pubertal female recipient mice. A small fragment of epithelial tissue from a donor mouse transplanted into the cleared fat pad of a pre-pubertal mouse will reconstitute an entire mammary gland under the stimulus of pubertal and pregnancy hormones. Transplantation of epithelial cell suspensions in sufficient numbers will also reconstitute a mammary gland. The identification of MaSCs (or committed progenitors) requires the transfer of purified populations of cells to identify which population has the greatest capacity to form mammary epithelium.
In previous studies, haematopoietic stem cells have been shown to lack lineage markers such as Ter119 (erythroid), CD3 and B220 (T and B lymphoid cells), Mac-1 (myeloid) and to express high levels of c-kit and Sca-1. Haematopoietic stem cells have also been shown to exclude the vital dye Hoechst33342 (Ho) with great efficiency, resulting in a side population (SP) in flow cytometric studies (Goodall et al., J Exp Med 183:1797-1806, 1996). Data using mammary epithelial cells that were propagated in vitro for several days and then purified by fluorescence-activated cell sorting (FACS) have found that Sca-1+ cells exhibit enhanced Ho dye exclusion and an enriched mammary repopulating capacity, suggesting that mammary stem cells reside within this population (Welm et al., Dev Biol 245:42-56, 2002). In addition, a SP has been identified in and purified from more freshly isolated mammary epithelial cell preparations, and found to be able to produce mammary epithelial structures on transplantation into MFPs (Alvi et al., Breast Cancer Res 5:R1-R8, 2003). However, in these studies MFP repopulation required large numbers (several thousand) of cells, and the comparative repopulating capacity of purified cell populations was not evaluated at limiting dilution. Furthermore, the purified cell populations in these studies were obtained from a source of cells that had been maintained in culture. These conditions are likely to modify cell surface marker phenotype and as such, the characteristics of the purified cells in these studies are unlikely to reflect those which exist in vivo.
There is a need, therefore, for a method of isolating a substantially homogenous population of MaSCs from a source of freshly isolated tissue.